Highly sensitive detection and characterization of minute quantities of chemicals and biochemicals represent desirable goals of modern analytical technologies. Robust molecular sensing devices would find uses in a wide range of biomedical, industrial, environmental, forensic, security and other contexts, for example, in the detection and identification of pathogens and chemicals at extremely low concentrations for disease diagnosis and environmental monitoring, in high throughput DNA sequencing and other genomics applications, and elsewhere.
Analytical methodologies have been described that employ intermolecular affinity binding interactions, typically non-covalent in nature, to detect binding or “capture” of an analyte of interest by a specific affinity ligand, for instance, including detection of bacterial, viral, parasitic or other microbial pathogens or pathogen-associated antigens, and detection of antibodies, cancer markers, and other analytes (e.g., Kittigul et al., Am J Trop Med Hyg. 1998 September; 59(3):352-6; Cordiano et al., J Immunol Methods. 1995 Jan. 13; 178(1):121-30; Olson et al., J Immunol Methods. 1990 Nov. 6; 134(1):71-9; Nerurkar et al., J Clin Microbiol. 1984 July; 20(1):109-14; Jia et al., J Virol Methods. 2009 October; 161(1):38-43; He et al., Clin Vaccine Immunol. 2007 May; 14(5):617-23; Xu et al., J Clin Microbiol. 2006 August; 44(8):2872-8; Che et al., J Clin Microbiol. 2004 June; 42(6):2629-35; Hunt et al., Brown et al., Am J Trop Med Hyg. 2001 September; 65(3):208-13; Loa et al., Avian Dis. 2000 July-September; 44(3):498-506; Lubenko et al., Transfus Med. 2000 September; 10(3):213-8; Chanteau et al., Int J Tuberc Lung Dis. 2000 April; 4(4):377-83; Brinker et al., J Clin Microbiol. 1998 April; 36(4):1064-9; Vyse et al., J Virol Methods. 1997 January; 63(1-2):93-101; Peterson et al., J Clin Microbiol. 1997 January; 35(1):208-12; Lairmore et al., AIDS Res Hum Retroviruses. 1993 June; 9(6):565-71; Heller et al., Vet Microbiol. 1993 October; 37(1-2):127-33; van Loon et al., Epidemiol Infect. 1992 February; 108(1):165-74; Wolf-Rogers et al., J Immunol Methods. 1990 Oct. 19; 133(2):191-8; Barsoum et al., Exp Parasitol. 1990 July; 71(1):107-13; Hierholzer et al., J Clin Microbiol. 1989 June; 27(6):1243-9; Hurley et al., J Immunoassay. 1986; 7(4):309-36; Wolff et al., Cancer Res. 53:2560-65 (1993); see generally, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; Weir, D. M., Handbook of Experimental Immunology, 1986, Blackwell Scientific, Boston, Mass.).
Beyond detection of the presence of an analyte following its involvement in an affinity binding interaction, sophisticated technologies are emerging that permit characterization of the analyte, often by comparing a single- or multiparameter physicochemical profile of the analyte to type-characteristic profiles generated using one or more known reference standards, and hence referred to as “fingerprinting” techniques (e.g., Li et al., Rapid Commun Mass Spectrom. 2009 23(22):3533-3542; Ali et al., J Agric Food Chem. 2009; Leski et al., Appl Environ Microbiol. Sep. 18, 2009; Weinkopff et al., J Parasitol. Jun. 18, 2009; Song et al., Proteomics. 2009 9(11):3090-9; Ortea et al., J Agric Food Chem. 2009 57(13):5665-72; Amini, Pharmeur Sci Notes. 2009(1):11-6; Shi et al., Biol Pharm Bull. 2009 32(1):142-6; Sun et al., J Chromatogr A. 2009 1216(5):830-6; Yin et al., Phytopathology. 2003 93(8):1006-13; Roy et al., Clin Cancer Res. 2008 14(20):6610-7; Pei et al., Zhongguo Zhong Yao Za Zhi. 2008 33(14):1662-8; Arthur, Methods Mol Med. 2008, 141:257-70; Zhao et al., Se Pu 2008 26(1):43-9; Woo et al., Anal Chem. 2008 80(7):2419-25; Damodaran et al., Genomics Proteomics Bioinformatics. 2007 5(3-4):152-7; Fellstrom et al., J Microbiol Methods. 2008 72(2):133-40; Song et al., Conf Proc IEEE Eng Med Biol Soc. 2006 1:4556-9; De Vuyst et al., Int J Food Microbiol. 2008 125(1):79-90).
The use of transmembrane channels has been demonstrated in stochastic analyte detection (Bayley et al., 2001 Nature 13:225-230), an electrochemical approach relying on the real-time observation of individual binding events between single substrate molecules and a receptor, as evidenced by altered (e.g., decreased or increased in a statistically significant manner) electrical conductance by the channel (receptor) as a result of substrate (analyte) binding. A wide range of processes, such as the transport of DNA, RNA, pharmaceutical agents, peptides, proteins, and polymers, have been studied by such approaches, for example, using electrophysiological measurements of individual membrane channels (Thieffry et al., 1988 EMBO J 7:1449; Hinnah et al., 2002 Biophys J 83:899; Alcayaga et al., 1992 FEBS Lett. 311:246-50; Benz et al., 1986 J Bacteriol 165:978; Movileanu et al., 2000 Nat. Biotechnol. 18:1091).
For instance, the transient blockade of ionic current through the Staphylococcus aureus alpha-hemolysin (α-HL) channel, a bacterial transmembrane pore-forming protein, has been used to measure the length of single-stranded DNA or RNA (Kasianowicz et al. Proc. Natl. Acad. Sci. USA 93, 13770-13773 (1996)). Subsequently, DNA hairpin molecules have been used to decelerate the DNA translocation rate through the alpha-hemolysin (α-HL) pore, to demonstrate the ability of a transmembrane ion channel to discriminate between single nucleotide polymorphisms (Vercoutere et al., 2001 Nat. Biotechnol. 19:248). Detection of base pair stacking and strand orientation within the pore have also been investigated (Vercoutere et al., 2003 Nucl Ac. Res. 31:1311; Howorka et al., 2001 Nat. Biotechnol. 19:636; deGuzman et al., 2006 Nucl. Ac. Res. 34:6425). The channel of α-HL with a covalently attached adapter molecule has been shown to discriminate the nucleotides A, T, G, and C (Clarke et al., 2009 Nat. Nanotechnol. 4:265).
The α-HL channel has also been reported to provide some ability to translocate small peptides (See e.g., Mohammad, et al., “Protein Sensing with engineered protein nanopores,” Methods Mol Biol. 2012, 870:21-37; Jetha, et al., “Nanopore Analysis of Wild-Type and Mutant Prion Protein (PrPc): Single Molecule Discrimination and PrPc Kinetics,” PLOS ONE, 2013, 8(2); Mereuta, et al., “Slowing down single-molecule trafficking through a protein nanopore reveals intermediates for peptide translocation,” Scientific Reports, 2014, 4:3885; Rosen, et al., “Single-molecule site-specific detection of protein phosphorylation with a nanopore,” Nat Biotechnol, 2014 February, 32(2):179-81); Movileanu, “Watching single proteins using engineered nanopores,” Protein Pept Lett. 2014 March, 21(3):235-46). However, at only about 1.2-1.5 nm, only a limited number of peptides are capable of being translocated through the α-HL channel and there is not an ability to differentiate between different peptides.
Other protein channels that have been investigated include alamethicin for detection of polyethylene glycol (Bezrukov, 2000 J Membr Biol. 174:1-13), and the reengineered MspA protein from M. smegmatis for translocation of ssDNA (Butler et al., 2008 Proc. Nat. Acad. Sci. USA 105:20647). Most studies involving nucleic acid transport through nanopores have focused on α-HL. However, the limiting lumen diameter of α-HL (about 1.2-1.5 nm) and other channels has restricted their DNA and RNA applications to translocation of single-stranded nucleic acid (Song, 1996 Science 274:1859). A similar limitation was also reported for the MspA nanopore (Butler et al., 2008).
In a small number of other membrane pore systems, evidence of double-stranded DNA (dsDNA) transport across the membrane has been presented (Szabo et al., 2002 Cell Physiol Biochem 12:127; Mobasheri et al. 2002 Eur J Bipohys 31:389; Carneiro et al., 2003 Biochim Biophys Acta 1612:144), but these systems are not robust and represent poor candidates for widespread use such as biomedical applications, due to their undesirable voltage gating properties and the associated signal fluctuation. For this reason, their potential is considered limited and researchers have switched instead to fabricating synthetic metal or silicon nanopores for potential use in DNA sequencing (Smeets et al, 2006 Nano Lett 6:89; Wang et al., 2001 Nat. Biotechnol. 19:622; lqbal et al., 2007 Nat. Nano 2:243). Such synthetic nanopores, however, suffer from shortcomings due to difficulties in reliably producing replicated structures having consistent properties from batch to batch, and also lack versatility with regard to the ability to engineer modifications to pore structures and/or to serve as substrates for modification by a wide range of chemical conjugation. As a result, the search for superior alternatives to currently available protein nanopores is still ongoing.
There is a need for improved compositions and methods that would provide a versatile membrane conductive channel platform for sensitively detecting and characterizing a wide range of analytes, having a lumen capable of accommodating dsDNA and polypeptides, that can be reliably and reproducibly assembled, that is not susceptible to voltage gating under working conditions, and that can be readily modified to feature a wide variety of specific affinity receptors for use in the detection and characterization of different analytes. The presently disclosed invention embodiments fulfill such a need, and offer other related advantages.